International Journal of

Molecular Sciences

Review

Biofilms in Endodontics—Current Status andFuture Directions

Prasanna Neelakantan 1,*, Monica Romero 2, Jorge Vera 3, Umer Daood 4, Asad U. Khan 5,Aixin Yan 6 and Gary Shun Pan Cheung 1

1 Discipline of Endodontology, Faculty of Dentistry, The University of Hong Kong, Pok Fu Lam,Hong Kong, China; spcheung@hku.hk

2 Department of Endodontics, Benemerita Universidad Autónoma de Puebla, Puebla 72000, Mexico;moniendo@gmail.com

3 Department of Postgraduate Endodontics, University of Tlaxcala, Private practice, Puebla 72420, Mexico;jveraro@yahoo.com.mx

4 Faculty of Dentistry, International Medical University, 126, Jln Jalil Perkasa 19,Bukit Jalil, 57000 Bukit Jalil, Malaysia; udaood@hotmail.com

5 Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit,Aligarh Muslim University, Aligarh, Uttar Pradesh 202001, India; asad.k@rediffmail.com

6 School of Biological Sciences, The University of Hong Kong, Pok Fu Lam, Hong Kong, China; ayan8@hku.hk* Correspondence: prasanna@hku.hk; Tel.: +852-2859-0581

Received: 20 July 2017; Accepted: 8 August 2017; Published: 11 August 2017

Abstract: Microbiota are found in highly organized and complex entities, known as biofilms,the characteristics of which are fundamentally different from microbes in planktonic suspensions.Root canal infections are biofilm mediated. The complexity and variability of the root canal system,together with the multi-species nature of biofilms, make disinfection of this system extremelychallenging. Microbial persistence appears to be the most important factor for failure of root canaltreatment and this could further have an impact on pain and quality of life. Biofilm removal isaccomplished by a chemo-mechanical process, using specific instruments and disinfecting chemicalsin the form of irrigants and/or intracanal medicaments. Endodontic research has focused on thecharacterization of root canal biofilms and the clinical methods to disrupt the biofilms in additionto achieving microbial killing. In this narrative review, we discuss the role of microbial biofilms inendodontics and review the literature on the role of root canal disinfectants and disinfectant-activatingmethods on biofilm removal.

Keywords: bacteria; disinfection; extracellular polysaccharide; irrigation; root canal; review

1. Introduction

A biofilm is a highly organized structure consisting of bacterial cells enclosed in a self-producedextracellular polymeric matrix attached on a surface. Biofilms may also be considered as a layer ofcondensation of microbiota or a microbial-derived community consisting of cells that are irreversiblyattached to a substratum or interface or to each other, and embedded in a matrix of extra-cellularpolysaccharides in addition to extracellular DNA (eDNA) and extracellular proteins [1,2]. In general,the exact composition varies with the microorganisms and nutrients available. The organisms in thebiofilms exhibit an altered phenotype with respect to growth rate and gene transcription [3,4].

Sessile bacterial cells (biofilm state) differ greatly from the free-floating bacterial cells (planktonicstate). The physiological properties of bacteria in biofilms are different compared to the same bacteriumin a culture media, partly because microorganisms in biofilms are protected from environmentalstresses by their matrix [5]. Microbes within the biofilm can be about 1000-fold more resistant towards

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antimicrobial agents and host defense mechanisms than their planktonic counterparts [6,7]. Bacterialcells grow more slowly in biofilms than in their planktonic state and therefore, take up antimicrobialagents more slowly [8]. The biofilm also provides a ready environment for mutation accumulation inmicrobial cells, further promoting their survival and persistence.

The process of root canal treatment involves enlarging the root canal with instruments andcleaning the space using chemical disinfectants to (i) remove remnant vital or necrotic tissues; (ii) kill themicrobiota within the root canal system, including disruption of the microbial biofilm and (iii) removethe accumulated hard tissue debris that is formed during root canal instrumentation [9,10]. In general,the aim of any disinfection strategy in healthcare is to reduce the bacterial load to a subcritical levelso that the patient’s immune response will allow healing. Root canal treatment is no different whereroot canal disinfection is considered the pivot of this therapy [11,12]. Interestingly, as elsewhere in thebody, the critical threshold that is compatible to health is unknown and hence endodontic researchhas focused on developing methods that can completely remove the bacterial biofilm, which is theideal goal.

Microbial biofilms in the root canal are highly resistant to disinfecting agents used in endodontictreatment. The complex and unpredictable nature of root canal anatomy and the multi-species biofilmsamplify the difficulty in eradication of the microbial biomasses from there [13,14]. The objective of thisreview is to discuss the microbiological aspects of root canal biofilms, clinical antibiofilm strategiesand research methods to study biofilms.

2. Microbiology of Root Canal Infections

The bacterial microflora of the root canal is initially dominated by aerobes and facultativeanaerobes [15,16]. As disease progresses, the ecology within the root canal system changes.Such changes maybe related to the oxygen tension when root canals are opened up duringtreatment, use of root canal irrigating agents and changes in the canal pH due to various materialsintroduced into the root canal. This results in phenotypic changes driven by genetic populationshift [17–19]. Endodontic infection may be primary or secondary. In general, primary infectioninvolves pulp inflammation and root canal infection following invasion by microbes or microbialby-products, eventually resulting in inflammation of the supporting tissues i.e., apical periodontitis.Secondary infection (or post-treatment infection) occurs either as reinfection (acquired or emergent),remnant (persistent) infection or recurrent infection (re-developed in teeth after apparent healing)in teeth that have been previously root canal treated [11]. Primary endodontic infections arepolymicrobial [19,20]. They are predominantly Bacteroides, Prophyromonas, Prevotella, Fusobacterium,Treponema, Peptostreptococcos, Eubacterium, and Camphylobacter species.

It is believed that persistence of microorganisms within the root canal system after treatment isthe major cause of treatment failure [21]. The ratios of the microbes in primary infections could bedifferent after root canal treatment, as well as a shift in species propagation and quantity. The microbialflora found in secondary infections, typically, are able to survive harsh conditions such as a widepH range and nutrient-limited conditions. There is a definite contrast in the microbial phenotypesin primary infections as compared to secondary infection, with the latter being predominatedby gram-positive bacteria [22–24]. Studies have shown the prevalence of certain species in teethwith post-treatment infection, such as Enterococci, Streptococci, Lactobacilli, Actinomyces and fungi(such as Candida). In particular, a high proportion of Enterococcus fecalis in cases with persistent apicalperiodontitis was noted [25,26].

Mixed infections are more common than single-organism isolates. Also, the wide variety oforganisms found in root canals can be partially related to the principal interests and culture techniquesof different investigators. Isolates from the exposed pulp are similar to the oral flora in whichgram-positive cocci predominate, and approximately 25% of the isolates are anaerobes. Organismsassociated with flare ups (which are emergency conditions characterized by pain and/or swelling)seem to share a similar composition as those from asymptomatic root canals [27,28]. These are usually

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phenotypic changes due to ecological shifts. Organisms cultured from infected canals elaborate avariety of invasive enzymes, but it is unclear if it can be equated with pathogenicity [29–31].

3. Characteristics of a Biofilm

Bacteria in the state of a biofilm are able to survive tough growth and environmental conditions,which, in part, is due to the protection offered by the extracellular matrix of the biofilms. This structureenables trapping of nutrients and allows metabolic cooperation among various resident bacteria of thesame or different species. The organized internal compartmentalization in a biofilm allows bacteria ofdifferent growth requirements to survive in their own microenvironments.

3.1. Protection of Biofilm Bacteria from Environmental Threats

Bacteria are capable of producing cell surface structures (e.g., capsule) or extracellular secretions(e.g., extracellular polysaccharide). The extracellular polysaccharide (EPS) can offer protection to allresident bacteria from various environmental stresses such as pH shifts, osmotic shock, UV radiationand desiccation [32,33]. It also alleviates the effect of any harmful substances that have to diffusethrough the EPS matrix before reaching the microorganisms.

3.2. Enhanced Tolerance to Antimicrobials

Long term use of drugs leads to the development of resistance among microorganisms dueto altered gene expression and transfer of resistance genes, rendering the antimicrobial agentineffective [32,34]. EPS matrix in bacterial cells acts as a barrier, trapping extracellular enzymessuch as β-lactamase and thus inactivates β-lactam antibiotics. Due to the depletion of nutrients,bacteria are forced into a dormant state and thereby protected from being killed readily [35].

Another mechanism responsible for antimicrobial tolerance is the selective location of anaerobicniches deep within the biofilm community, as oxygen can be completely depleted by bacteria atthe biofilm surface [36]. A sub-population known as persisters is often found within the biofilmcommunity. These persisters can belong to any bacterial type and are in a highly resistant phenotypicstate that is resistant to killing by many antimicrobial agents [37,38].

3.3. Quorum Sensing

Quorum sensing is a bacterial cell-to-cell communication system [39]. Using chemical signalingmolecules, bacteria are able to communicate with one another. For example, molecules such as thecompetence-stimulating peptide (CSP) are important to co-ordinate gene expression. Quorum sensingallows bacteria to monitor the environment for other bacteria and allow alteration of one’s behavior ina population-wide scale. Microorganisms function less effectively as individuals compared to coherentgroups [40]. Pooling the activity of a quorum of cells can improve the successful persistence of bacteria.

Quorum sensing is known to be involved in the formation of biofilms and to cope withenvironmental stresses [41]. The characteristics of a specific strain of microbe determine its abilityto co-exist. Such an association has been demonstrated between E. fecalis, Streptococcus gordonii andLactobacillus salivarius [42]. One reason for this could be the differential starvation endurance of E. fecalisin mono-species and multi-species biofilms, depending on which microbe it is in association with. Theprotease production in biofilm environments appears to have an influence on the ability of co-existencebetween bacteria, as it is related to the virulence of bacteria [42]. Specific quorum sensing geneshave also been implicated in the biofilm forming ability of E. fecalis (e.g., S-ribosylhomocysteine lyase[luxS]). LuxS encodes autoinducer 2 which is considered a cross-species signaling molecule [43]. It issurprising however, that quorum sensing and its inhibition have not been well studied within theroot canal system. Future research should focus on using quorum-sensing inhibitors in root canalantibiofilm strategies.

Figures 1 and 2 show some multi-species biofilms from the apical foramen of root canalsdemonstrating communicating channels, fusion of extracellular fibers, compact extracellular polymers

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and their network. Figure 3 shows a mono-species biofilm of Enterococcus fecalis, with its denseEPS matrix.

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polymers and their network. Figure 3 shows a mono-species biofilm of Enterococcus fecalis, with its dense EPS matrix.

(i) (ii)

Figure 1. Scanning electron microscopic images of the apical of the apical foramen of a tooth showing a mature biofilm of (i) Leuconostoc spp. (10,000×). A multilayered polymeric matrix is evident. Also noticed are communicating channels in the biofilm (A), extracellular fibers secreted by bacterial cells (B), metabolically active cells characterized by cell division (C) and compact extracellular polymers (D); (ii) Biofidobacterium spp. in a maturation state (10,000×). Secretion of polymers in granular form by the bacterial cells (A), microfilaments of extracellular polymers surrounding the rod shape of the microorganism (B) and extracellular polymers in the maturation state (C) can be seen (Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González, UASLP Mexico).

(i) (ii)

Figure 2. Scanning electron microscopic images of (i) Clostridium botulinum and Streptococcus anginosus (5000×). Communication channels inside the biofilm (A), fusion of extracellular fibers in laminar shape (B) and the presence of numerous polymeric fibers secreted by rods and cocci forming a crisscross pattern (C) are evident. (Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González, UASLP Mexico); (ii) 3 weeks biofilm of Enterococcus fecalis on dentin (2000×). The dense EPS matrix is visible and the challenge in eradicating biofilms of this microbe is, in part, attributed to the ability of medicaments to penetrate this matrix. This is the most commonly implicated bacterium in root canal treatment failure.

Figure 1. Scanning electron microscopic images of the apical of the apical foramen of a toothshowing a mature biofilm of (i) Leuconostoc spp. (10,000×). A multilayered polymeric matrix isevident. Also noticed are communicating channels in the biofilm (A), extracellular fibers secreted bybacterial cells (B), metabolically active cells characterized by cell division (C) and compact extracellularpolymers (D); (ii) Biofidobacterium spp. in a maturation state (10,000×). Secretion of polymers ingranular form by the bacterial cells (A), microfilaments of extracellular polymers surrounding the rodshape of the microorganism (B) and extracellular polymers in the maturation state (C) can be seen(Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González, UASLP Mexico).

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mers and their network. Figure 3 shows a mono-species biofilm of Enterococcus fecalis, with its dense EPS matrix.

(i) (ii)

Figure 1. Scanning electron microscopic images of the apical of the apical foramen of a tooth show-ing a mature biofilm of (i) Leuconostoc spp. (10,000×). A multilayered polymeric matrix is evident. Also noticed are communicating channels in the biofilm (A), extracellular fibers secreted by bacteri-al cells (B), metabolically active cells characterized by cell division (C) and compact extracellular polymers (D); (ii) Biofidobacterium spp. in a maturation state (10,000×). Secretion of polymers in granular form by the bacterial cells (A), microfilaments of extracellular polymers surrounding the rod shape of the microorganism (B) and extracellular polymers in the maturation state (C) can be seen (Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González, UASLP Mexi-co).

(i) (ii)

Figure 2. Scanning electron microscopic images of (i) Clostridium botulinum and Streptococcus angino-sus (5000×). Communication channels inside the biofilm (A), fusion of extracellular fibers in laminar shape (B) and the presence of numerous polymeric fibers secreted by rods and cocci forming a criss-cross pattern (C) are evident. (Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González, UASLP Mexico); (ii) 3 weeks biofilm of Enterococcus fecalis on dentin (2000×). The dense EPS matrix is visible and the challenge in eradicating biofilms of this microbe is, in part, attributed to the ability of medicaments to penetrate this matrix. This is the most commonly implicated bacte-rium in root canal treatment failure.

Figure 2. Scanning electron microscopic images of (i) Clostridium botulinum and Streptococcus anginosus(5000×). Communication channels inside the biofilm (A), fusion of extracellular fibers in laminar shape(B) and the presence of numerous polymeric fibers secreted by rods and cocci forming a crisscrosspattern (C) are evident. (Courtesy: Dr. Ana Maria González Amaro, Maria Verónica Méndez González,UASLP Mexico); (ii) 3 weeks biofilm of Enterococcus fecalis on dentin (2000×). The dense EPS matrix isvisible and the challenge in eradicating biofilms of this microbe is, in part, attributed to the ability ofmedicaments to penetrate this matrix. This is the most commonly implicated bacterium in root canaltreatment failure.

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Figure 3. Confocal laser scanning microscopic (CLSM) image (single slice at 20× magnification) of a 1 week biofilm of Enterococcus fecalis within the root canal. Dense aggregates of bacteria are seen within the dentinal tubules. The advantage of CLSM imaging is to detect the presence of apparently dead (red) and apparently live (green) bacteria. It is also possible to perform 3 dimensional reconstruction of Z-stack images to study biofilm architecture.

4. Methods of Studying Biofilms in Endodontics—Concepts from the Non-Endodontic World

Contemporary microbiological research has helped us develop an understanding that interactions between microorganisms are complex and play an important role in pathogenesis, either by synergistic or antagonistic mechanisms. This understanding has led to increased interest in studying the mechanisms involved and to a shift away from being monomicrobial to polymicrobial nature, when studying root canal biofilms in vitro [44,45]. Recent studies have focused on multi-species bacterial biofilms as a more realistic reflection of what happens in the real in vivo situation in the root canal system. Endodontic infection demonstrates significant microbial diversity, and the properties of such complex biofilms cannot be observed in studies with one species alone [4,46–48].

It is known that the functional properties of biofilms are intimately related to their 3-dimensional structure. It is necessary to understand and to study this architecture, not only to observe them on a cellular scale, but also in relation to the extracellular matrix, which creates such a special ecosystem. To achieve this objective, it is necessary to use techniques that allow observation of reconstructions of the communities [49].

Appreciation of bacteria living in multi-species biofilms rather than in mono-species communities, or in a free-living state, has changed the way bacteria are studied in the laboratory. Models are being created to replicate biofilm environments [50,51], thereby entering times of re-learning bacterial behavior as they live and survive in biofilms. In the same way, meta-genomic studies have increased knowledge of the complexity of microbial relations within the biofilm population [31,52].

Nowadays, biofilms are studied in two ways: in one, the consortia of microorganisms as a single unit and, in the second, by studying the effects and relationships between one species and others [44]. Because of advances in technology and computational biology, it is possible to study gene and protein expression in such communities, thus revealing the role that each species has in that specific community [53]. Pragmatically speaking, identification of the exact nature of an intracanal biofilm is a real challenge, because few techniques are capable of re-creating both the extracellular matrix as well as the microorganisms in that biofilm.

There are numerous advantages of using an in vitro biofilm model, which include ease of modification if necessary, control of variables, low cost and ease of replication. They are also very useful in answering some initial but fundamental questions, providing preliminary data, which is essential for future confirmation by in vivo testing.

Figure 3. Confocal laser scanning microscopic (CLSM) image (single slice at 20× magnification) of a1 week biofilm of Enterococcus fecalis within the root canal. Dense aggregates of bacteria are seen withinthe dentinal tubules. The advantage of CLSM imaging is to detect the presence of apparently dead(red) and apparently live (green) bacteria. It is also possible to perform 3 dimensional reconstruction ofZ-stack images to study biofilm architecture.

4. Methods of Studying Biofilms in Endodontics—Concepts from the Non-Endodontic World

Contemporary microbiological research has helped us develop an understanding that interactionsbetween microorganisms are complex and play an important role in pathogenesis, either by synergisticor antagonistic mechanisms. This understanding has led to increased interest in studying themechanisms involved and to a shift away from being monomicrobial to polymicrobial nature,when studying root canal biofilms in vitro [44,45]. Recent studies have focused on multi-speciesbacterial biofilms as a more realistic reflection of what happens in the real in vivo situation in the rootcanal system. Endodontic infection demonstrates significant microbial diversity, and the properties ofsuch complex biofilms cannot be observed in studies with one species alone [4,46–48].

It is known that the functional properties of biofilms are intimately related to their 3-dimensionalstructure. It is necessary to understand and to study this architecture, not only to observe them on acellular scale, but also in relation to the extracellular matrix, which creates such a special ecosystem.To achieve this objective, it is necessary to use techniques that allow observation of reconstructions ofthe communities [49].

Appreciation of bacteria living in multi-species biofilms rather than in mono-species communities,or in a free-living state, has changed the way bacteria are studied in the laboratory. Models arebeing created to replicate biofilm environments [50,51], thereby entering times of re-learning bacterialbehavior as they live and survive in biofilms. In the same way, meta-genomic studies have increasedknowledge of the complexity of microbial relations within the biofilm population [31,52].

Nowadays, biofilms are studied in two ways: in one, the consortia of microorganisms as asingle unit and, in the second, by studying the effects and relationships between one species andothers [44]. Because of advances in technology and computational biology, it is possible to studygene and protein expression in such communities, thus revealing the role that each species has in thatspecific community [53]. Pragmatically speaking, identification of the exact nature of an intracanalbiofilm is a real challenge, because few techniques are capable of re-creating both the extracellularmatrix as well as the microorganisms in that biofilm.

There are numerous advantages of using an in vitro biofilm model, which include ease ofmodification if necessary, control of variables, low cost and ease of replication. They are also veryuseful in answering some initial but fundamental questions, providing preliminary data, which isessential for future confirmation by in vivo testing.

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4.1. Microtiter Plate-Based Systems

These are useful and consistently used biofilm model systems. The system is closed; therefore,there is no flow in or out of the reactor during experimentation. Consequently, the environmentin the experimental model changes, for example, with regard to the availability of nutrients andmolecules [54]. The microtiter plate-based system is used to perform different tests at the same time,which may be ideal for rapid screening of methods for biofilm disinfection and removal [55]. Biofilmquantification with microtiter plates may be categorized into biomass assays, viability assays andmatrix quantification assays [56].

Biofilm analysis on microtiter-plate based systems may be performed using crystal violet, nucleicacid stains such as Styo9, non-fluorescent fluorescein diacetate, tetrazolium salts such as XTT, resazurinor dimethyl methylene blue. These methods show differential results for fungal and bacterial biofilms.Furthermore, it has been suggested that the Styo9 assay should not be used for CFU measurementsin biofilms and that the crystal violet assay was non-repeatable for Pseudomonas aeruginosa biofilms(56). A limitation of the Styo9 assay is that it depends on microbial cell wall integrity, and thuscan be misrepresented by microbes that may be dead, yet have an intact cell wall. CFU countsonly reproducing cells, and can over-quantify killed cells based on reduction of metabolic activity(chemostatic treatments would be considered killed). Since these test methods measure differentanalytics to describe viability, it is quite possible the results do not match. Hence, it may be preferableto perform tests such as FDA or resazurin for quantification of biofilms with differentiation betweendead and live cells [56].

4.2. Flow Displacement Biofilm Model Systems

In contrast to the microtiter plate-based system, the flow displacement system is open. In thissystem, the growth medium containing the nutrients needed for growth is added at a constant rate,with the waste products and toxins from the biofilm removed simultaneously [57]. The concept of flowdisplacement is based on the premise that an initial film of macromolecular components needs to formon a surface to allow microbial adhesion [57]. The fluid flow, in an optimal manner ensures adhesionof microbial cells to a substrate, which is a characteristic property of any biofilm.

It has been suggested that experiments using parallel plate flow chamber performed withcontrolled hydrodynamic conditions would offer ideal flow rates for microbial adhesion and reducestechnical variables [58]. This also helps in repeatability of the study designs in different laboratories.

4.3. Modified Robbins Device

The modified Robbins device is a device in which there is a continuous formation of biofilm whichis exposed to fluid flow [59]. This device may employ silicone or hydroxyapatite discs as the substratefor biofilm growth, with or without addition of agents that support or inhibit microbial growth. Themain advantage of this system is that it allows evaluation of more than one antibiofilm agent in thesame experiment [60]. This device can also be modified and used in conjunction with flow devices.

4.4. Microfluidic Device

This is becoming a popular method of studying biofilms, because of the possibility of forminga biofilm under conditions similar to that physiologically, for example, cell-to-fluid volume ratiosand flow velocities. Also, since the chamber is small, it allows for a single cell resolution analysisof the biofilm under tightly controlled conditions [61]. In essence, this device allows the analysis ofchemical assays using small quantities of liquids on a small chip. Such an approach could be veryuseful in techniques such as polymerase chain reaction, protein analysis and DNA sequencing [62,63].However, there are challenges in this approach in terms of analysis of biofilms, with specific referenceto quantification using methods such as fluorescent staining [64]. An interesting development inthis aspect was the application of a modified confocal reflection microscopic approach, termed

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the continuous optimizing confocal reflection microscopy, to quantitatively study the biovolumeof biofilms [64].

In essence, various techniques and technologies have helped to increase our understandingof biofilm physiology and the behavior of bacteria living there. However, these experiments areperformed on in vitro biofilm models that may not accurately represent or mimic in vivo biofilmbehavior or they simply under-represent it. Another important issue is that in vitro models lack thereaction or challenge imposed on a biofilm in vivo due to the host immune response, therefore limitingthe knowledge of how this very important aspect would add to the survival and/or elimination ofa biofilm.

4.5. Confocal Laser Scanning Microscopy

In recent years, confocal laser scanning microscopy has emerged as a very good method to studybiofilms structure [65], because it allows non-destructive investigation of these ecosystems and thehydrated spatial arrangement at cellular scale. The use of fluorescent markers allows targeting ofparticular cells or, even certain components of the extracellular matrix.

The use of specific stains [e.g., live/dead stains] allows differentiation of live and dead bacteriaoften times demonstrated by green or red fluorescent signals [66,67] (Figure 3). However, this may becontroversial as the processing of a specimen may result in bacterial death and hence falsely reportedas efficacy of a treatment approach. Nevertheless, this technique allows us to have a fair idea ofthe effectiveness of irrigating solutions and techniques in bringing about disruption of the biofilmstructure using a three-dimensional reconstruction of the biomass as to study biofilm architecture.Limitations are the same as those for light microscopy, including the fact that it is not able to visualizethe cellular ultrastructure, which requires higher-resolution imaging.

4.6. Fluorescent Microscopic Techniques with Super Resolution

New microscopic techniques such as STED (Stimulated Emission Depletion), PALM(Photo-Activated Localization Microscopy) and SIM (Structured-Illumination Microscopy) are able toeliminate some of the limitations in terms of resolution [68]. However, for this purpose, the biofilmmust be labeled with fluorescing dyes. Hence, it is essential to identify specific markers for the biofilmcomponents that are to be imaged and analyzed.

4.7. Scanning Electron Microscopy (SEM)

The use of SEM overcomes some of the limitations related to the use of fluorescent dyes. Thistechnique allows scanning of microbial ecosystems to obtain qualitative information as well as detailedanalysis of morphological structures, such as the cellular surface of one bacterium or identification ofdamage to the cell membrane. It also allows analysis of cell-to-cell interactions by detecting structuralchanges in the ecosystems [69,70]. However, it must be appreciated that the sample preparation forthis kind of microscopy usually involved high vacuum conditions which results in distortion of theextracellular polymeric matrix [71]. Low vacuum methods, such as environmental SEM, may be moreuseful in this regard.

5. Removal of Endodontic Biofilms during Root Canal Treatment

The objectives of root canal irrigation are to dissolve vital or necrotic pulp tissues, disruptendodontic biofilms, neutralize endotoxins and remove the smear layer. Antimicrobial activity andbiofilm destruction appear to be the most important objectives targeted towards the etiology of pulpand periradicular infections. Figure 4 show the presence of tissue debris within microbial biofilms inthe isthmus between canals in a molar tooth.

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Figure 4. Histological section of the isthmus area between two canals in a mandibular molar, stained by Taylor modified Brown and Brenn stain (16× and 100×) showing the presence of numerous bacterial masses with tissue. A higher magnification (100× and 400×) reveals the presence of residual bacteria and debris in the communications between canals after cleaning and instrumentation of root canal systems. This is the existing challenge in root canal treatment (Courtesy: Dr. Domenico Ricucci, Italy).

The challenge in endodontic treatment is for the disinfectants to reach those minute areas and facilitate removal of the inflamed or necrotic tissue within biofilms. This section of the review will focus on reports concerning biofilm destruction by the most commonly used root canal irrigants. In line with the contemporary understanding of endodontic biofilms, only studies that focus on antibiofilm strategies (rather than just antimicrobial activity) are discussed.

5.1. Root Canal Irrigants

5.1.1. Proteolytic Irrigants

Sodium hypochlorite (NaOCl) is regarded as the most potent disinfectant in endodontics due to its excellent ability to dissolve vital and necrotic tissues, in addition to its antimicrobial activity [10,72,73]. In endodontic therapy, NaOCl is used in concentrations ranging from 0.5 to 6%, all of which demonstrate antibacterial activity [74–76]. Studies show that the antimicrobial activity is not concentration dependent, but tissue dissolution and biofilm disruption are concentration dependent [77,78]. The recommended irrigation regimen involves a sequential use of NaOCl and a decalcifying agent. Ozdemir et al., concluded that the combined application of 17% EDTA and 2.5% NaOCl reduces the amount of intracanal biofilm significantly [79]. The effectiveness of sodium hypochlorite may be improved by warming the solution, use of agitation/activation methods, increasing the volume of the irrigant, and lowering the pH of the irrigant solution [13,80,81]. While the role of warm NaOCl is not clear, owing to the fact that extreme temperature is rapidly buffered within the root canal system [82], it could be advantageous to continuously deliver warm NaOCl via new devices based on negative pressure [83]. Alternatively, techniques such as ultrasonic activation also increases the temperature of the irrigating solution and may prove to be beneficial [81]. The endodontic literature is consistent in demonstrating that NaOCl is able to completely disrupt the biofilms within the root canal system. However, Rosen et al., reported a very interesting finding that NaOCl induces a viable but non-culturable state of bacteria in biofilms and that this might contribute to bacterial persistence [84].

5.1.2. Antiseptics

Chlorhexidine (CHX) gluconate with a very broad antimicrobial spectrum is used as an oral antiseptic mouthwash for plaque control and as an irrigant for periodontal therapy and infected root canals [72,85]. It has a lower grade of toxicity compared to sodium hypochlorite and sustained action i.e., substantivity. A concentration of 2% is recommended as a root canal irrigant [10,86].

Figure 4. Histological section of the isthmus area between two canals in a mandibular molar, stained byTaylor modified Brown and Brenn stain (16× and 100×) showing the presence of numerous bacterialmasses with tissue. A higher magnification (100× and 400×) reveals the presence of residual bacteriaand debris in the communications between canals after cleaning and instrumentation of root canalsystems. This is the existing challenge in root canal treatment (Courtesy: Dr. Domenico Ricucci, Italy).

The challenge in endodontic treatment is for the disinfectants to reach those minute areas andfacilitate removal of the inflamed or necrotic tissue within biofilms. This section of the review willfocus on reports concerning biofilm destruction by the most commonly used root canal irrigants. In linewith the contemporary understanding of endodontic biofilms, only studies that focus on antibiofilmstrategies (rather than just antimicrobial activity) are discussed.

5.1. Root Canal Irrigants

5.1.1. Proteolytic Irrigants

Sodium hypochlorite (NaOCl) is regarded as the most potent disinfectant in endodontics dueto its excellent ability to dissolve vital and necrotic tissues, in addition to its antimicrobialactivity [10,72,73]. In endodontic therapy, NaOCl is used in concentrations ranging from 0.5 to6%, all of which demonstrate antibacterial activity [74–76]. Studies show that the antimicrobialactivity is not concentration dependent, but tissue dissolution and biofilm disruption are concentrationdependent [77,78]. The recommended irrigation regimen involves a sequential use of NaOCl anda decalcifying agent. Ozdemir et al., concluded that the combined application of 17% EDTA and2.5% NaOCl reduces the amount of intracanal biofilm significantly [79]. The effectiveness of sodiumhypochlorite may be improved by warming the solution, use of agitation/activation methods,increasing the volume of the irrigant, and lowering the pH of the irrigant solution [13,80,81]. While therole of warm NaOCl is not clear, owing to the fact that extreme temperature is rapidly buffered withinthe root canal system [82], it could be advantageous to continuously deliver warm NaOCl via newdevices based on negative pressure [83]. Alternatively, techniques such as ultrasonic activation alsoincreases the temperature of the irrigating solution and may prove to be beneficial [81]. The endodonticliterature is consistent in demonstrating that NaOCl is able to completely disrupt the biofilms withinthe root canal system. However, Rosen et al., reported a very interesting finding that NaOCl inducesa viable but non-culturable state of bacteria in biofilms and that this might contribute to bacterialpersistence [84].

5.1.2. Antiseptics

Chlorhexidine (CHX) gluconate with a very broad antimicrobial spectrum is used as an oralantiseptic mouthwash for plaque control and as an irrigant for periodontal therapy and infected rootcanals [72,85]. It has a lower grade of toxicity compared to sodium hypochlorite and sustained actioni.e., substantivity. A concentration of 2% is recommended as a root canal irrigant [10,86]. Arias-Moliz

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et al., showed that alternating the application of CHX and cetrimide resulted in a higher percentagereduction of Enterococcus fecalis compared to the combined use of these 2 agents [87]. Cetrimidefacilitates the destruction of EPS matrix allowing CHX to act more directly on Enterococcus fecalis thusresulting in a greater bactericidal potential [88].

In two different studies, Baca et al., concluded that the combination of 2% CHX and 0.2%cetrimide as a final irrigating solution showed maximum residual and antimicrobial activity onEnterococcus fecalis biofilm [88,89]. CHX Plus, which is a combination of chlorhexidine gluconatewith surface modifiers, showed higher levels of bactericidal activity compared to CHX alone [90].Mechanical agitation of CHX has been proven to promote its antimicrobial effectiveness. ComparingCHX and NaOCl, it is now known that although CHX exhibits antibacterial activity but is unable todestroy the biofilm structure [91,92].

Another bisbiguanide, Alexidine (ALX) was introduced as a root canal irrigant quite recently.Alexidine differs from CHX due to the presence of 2 hydrophobic ethylhexyl groups, which enablesrapid antibacterial action. Compared to CHX, ALX has a greater affinity for lipoteicoic acids resultingin an increased permeability into the bacterial membrane [93]. ALX (1%) has been shown to bringabout bacterial killing similar to 2% CHX, although both agents do not appear to disrupt the biofilmsof E. fecalis [94,95]. Octenidine hydrochloride (OCT) is a positively charged bispyridinamine. It acts bybinding to the negatively charged bacterial cell envelope resulting in disruption of the functions ofbacterial cell membrane [96]. The antimicrobial activity of OCT on Candida albicans was studied byEldeniz and coworkers who reported that OCT was able to totally eliminate all Candida cells whenused as a root canal irrigant [97]. Not enough evidence exists at this time evaluating alexidine oroctendine on complex biofilms and future research is warranted.

Iodine potassium iodide (IKI) was first used by a French physician, Lugol to treat scrofula in1829. In 1927, iodine products were initially used as root canal irrigants. IKI has a broad spectrum ofantimicrobial action and is effective against enteric bacteria, enteric virus, and protozoan cysts. Due toits lack of tissue dissolving capabilities, it has been suggested to use IKI after canal instrumentationand irrigation with sodium hypochlorite [98]. Wang et al., found that the addition of a detergent toIKI increases the antibacterial effects against Enterococcus fecalis in the dentinal tubules [99]. However,the literature lacks evidence on the effectiveness of this compound on the biofilm structure itself.

5.1.3. Demineralizing Agents

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent recommended as an adjuvant inroot canal therapy. Many authors have shown its efficacy for removing the inorganic portion of thesmear layer [73]. However, EDTA has little or no antimicrobial activity. Alternating the use of NaOCland EDTA during root canal treatment appears to be a promising approach to remove the organicand inorganic debris, in addition to disrupting microbial biofilms [81]. Soares et al., studied theeffectiveness of chemomechanical preparation with alternating use of sodium hypochlorite and EDTAon an intracanal E. fecalis biofilm and found that the alternating use of these 2 agents promoted theelimination of root canal E. fecalis biofilm [100]. While EDTA has been shown to be effective againstCandida albicans [101], its efficacy on Candida albicans biofilms or multi-species biofilms has not yetbeen documented.

Another demineralizing agent, maleic acid has been shown to be effective against E. fecalis ata concentration of 0.88% for 30 seconds [102]. It is believed that the cell membrane permeability isaltered due to the decrease in the internal pH of the microbial cell, resulting in death of the bacterialcell. However, such an action was not shown against intra-orally formed multispecies biofilms [103].A 2.25% peracetic acid (PAA) solution was recommended as a final irrigant after the use of sodiumhypochlorite during instrumentation [104]. Peracetic acid has been shown to be more effective thanchlorhexidine against root canal mono-species E. fecalis biofilms [91,103]. One may consider peraceticacid as a single irrigant with two purposes—it is a demineralizing agent with strong antibacterialproperties [104].

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5.1.4. Combination of Irrigating Solutions

MTAD (BioPure MTAD, Dentsply Sirona Endodontics, York, PA, USA) is a mixture of 3%doxycycline, 4.25% citric acid and 0.5% Tween 80. Prabhakar and coworkers showed completeinhibition of bacterial growth by MTAD in a 3 week old biofilm [105]. In contrast, some studies haveconcluded that MTAD did not have good antibacterial activity against E. fecalis [103,106]. QMiX is amixture of CHX, EDTA and a detergent. It has been shown to be as effective as NaOCl and superior toCHX against Enterococcus fecalis and mixed plaque bacteria in planktonic and biofilm states [107,108].

An interesting concept called continuous chelation involves mixing 5% sodium hypochlorite with18% etidronic acid to serve as a single proteolytic-antibacterial-demineralising solution [104]. Etidronicacid is a weak chelator and hence, when mixed with NaOCl, can be indicated as an irrigant duringthe entire instrumentation process. The continuous chelation protocol has been shown to bring aboutexcellent antibiofilm activity against biofilms of E. fecalis [81,91].

5.1.5. Natural Agents (Phytotherapeutic or Ethnopharmacological Approaches)

Although several studies have evaluated the antibacterial actions of essential oils, none have testedthem on biofilm models. Hence, this category of materials is not discussed. Other phyotherapeuticagents such as Berberine, Morinda citrifolia and curcumin have also been evaluated against root canalbiofilms. Berberine, an antimicrobial plant alkaloid, when combined with 1% chlorhexidine hasantibacterial activity comparable to 5.25% sodium hypochlorite and 2% chlorhexidine [109]. It hasbeen tested on Candida albicans biofilms in a non-endodontic model, and appears to show favorableantibiofilm activity when combined with miconazole [110].

Curcumin is a naturally occurring yellow compound found in Curcuma longa L. which ismost commonly known as turmeric. Many studies have shown that curcumin has antimicrobial,anti-inflammatory and anti-oxidant activities. Recent evidence shows that curcumin is an effectivephotosensitizer and brings about antibiofilm activity and dentinal tubule disinfection similar to sodiumhypochlorite [6,111].

5.1.6. Nanoparticles Based Disinfection

In recent years, the use of nanoparticles to disinfect root canals has gained popularity dueto their broad spectrum antibacterial activity. Chitosan (CS-np), zinc oxide (ZnO-np) and silver(Ag-np) nanoparticles possess a broad spectrum of antimicrobial activity, caused by altering cell wallpermeability resulting in cell death [112–114]. This topic has been excellently reviewed by Shresthaand Kishen recently [113].

Rose bengal-functionalized CS-np have been widely studied and appear to be effectiveagainst monospecies and multispecies biofilms, even in the presence of tissue inhibitors [115].When attempting to disinfect the root canal system, the priority is on targeting the microbial biofilmand to render the substrate less or not amenable to microbial adhesion. However, it is also importantto protect the substrate dentin from further degradation or, at least, not damaged by the irrigants interms of their physical and chemical structure [116,117].

Photodynamic therapy (PDT) has been used to disinfect root canals because it has antimicrobialactivity and the ability to cross-link collagen with proteins. Rose bengal, a non-toxic dye, becomescytotoxic when activated with a low-intensity visible light and oxygen, targeting cells or tissues ingeneral as well as the lesion to which it is directed [117,118]. Chitin, obtained from crustaceans, such ascrabs and shrimps, is another important polymer. From this, chitosan, which is biodegradable andnon-toxic, can be derived and used for biomedical and pharmaceutical applications [113,119]. Chitosancan also be decorated with photosensitizers [120,121] and chitosan conjugated with rose bengal hasbeen reported to enhance the degradation resistance of collagen than rose bengal alone, lacks residualactivity, and is stable in the environment [115,118].

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Silver nanoparticles sized 10–100 nm was demonstrated to possess powerful antibacterialactivity against gram-positive and gram-negative bacteria [114]. Furthermore, mesoporous bioactivecalcium silicate nanoparticles and bioactive glass powder loaded with AgNp demonstratedsignificant reduction in adhesion of E. fecalis biofilms and this was further exemplified by ultrasonicactivation [122]. These materials appear to be potentially useful intracanal disinfectants and furtherresearch is needed on the biofilm disruption by these materials.

Nanoparticles with reactive molecules and nanoscale materials have the potential tocombat microorganism resistance, since they have the advantages of very small sizes, a largesurface-area-to-mass ratio and very good reactivity [123]. PDT used together with nanoparticleshas enhanced the efficacy of treatment against microorganisms like bacteria by improving deliveryand reducing activation [124]. However, some limitations exist; they can form some aggregatescompromising the area where they are used [125] and, because of the anatomical complexities present inthe root canal system, effective delivery to target areas may not be possible. Future research must focuson the delivery of nanoparticles into all corners of the root canal system to enable optimal disinfection.

5.1.7. Miscellaneous Interventions

Enzymatic irrigation was introduced by Niazi and coworkers, who evaluated the effectivenessof 1% trypsin and 1% proteinase K, with or without ultrasonic activation, on a multi-species biofilm.Trypsin with ultrasonic activation was able to effectively kill both aerobic and anaerobic bacteria andhas the capability of disrupting the biofilm [126].

Agents that interfere with the cell wall, such as D-amino acids, specifically D-leucine has beendemonstrated to bring about efficient dispersal of Enterococcus fecalis biofilms. It has been suggestedthat the dispersal of biofilms by sub-toxic concentrations of this agent reduces the success of resistantorganisms [84]. Table 1 summarizes of the role of each category of agents in root canal disinfection.

Table 1. Summary of effect of commonly used root canal disinfectants on bacterial suspensions orbiofilms in an endodontic disinfection model.

Antimicrobial Agent Chemical TypeConcentration

Used/Recommended inRoot Canal Disinfection

Activity on BacterialSuspensions (Root Canal

Models Only)

ACTIVITY onMono-Species or

Multi-Species Biofilms(Endodontic Taxa Only)

Sodiumhypochlorite (NaOCl) Halogen releasing agent 1–6% Yes Yes

Chlorhexidine (CHX) Bisbiguanide 2% Yes Unclear

Alexidine (ALX) Bisbiguanide 1–2% Yes Unclear

Octenidine (OCT) Bisbiguanide Yes Unclear

Iodine PotassiumIodide (IKI) Halogen releasing agent 2–5% Yes Insufficient evidence

Ethylene diaminetetraacetic acid (EDTA) Polyprotic acid 15–17% No No

Maleic acid Diprotic acid 7% Yes Insufficient evidence

Peracetic acid Organic peroxide 2.25% Yes Yes

MTAD Mixture of antibiotic,organic acid (citric acid), detergent Yes Unclear

QMix Mixture of CHX and EDTA Yes Yes

Etidronic acid(with 6% NaOCl) Bis-phosphonate 18% Yes Yes

Curcumin Phyto-polylphenol — Yes Yes

Chitosan with RoseBengal

Polysaccharide withphotosensitiser Yes Yes

Silver nanoparticles Metallic nanoparticle Yes Yes

Trypsin and ProteinaseK Enzymes 1% Yes Yes (Trypsin)

D-leucine Amino acid Yes Yes

The term “unclear” has been used when methods other than confocal laser scanning microscopy have been used todetect the effect on biofilms.

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5.1.8. Intracanal Medicaments

Intracanal medicaments have conventionally been recommended as root canal dressing betweenappointments. However, with the increasing trend towards single visit endodontics, and the lack of aclear advantage of multiple visit treatments over single visit [127–129], the role of inter-appointmentdressings is questionable and may wane with time. Nevertheless, considering the present evidencebase (or lack of, thereof) to support one approach over the other, this section will briefly focus on therole of intracanal medicaments in root canal disinfection. The reader is referred to other reviews onthis topic for details on the composition and mechanisms [130,131]. A systematic review on the effectof intracanal medicaments on bacterial biofilm concluded that these agents had limited action againsta biofilm [132]. Furthermore, even if these agents are able to bring about bacterial killing inside abiofilm, it is unknown if they can disrupt or remove biofilms from the root canal system.

Calcium hydroxide has been shown to be ineffective against biofilms of E. fecalis even after24 hours of treatment [133]. It was also reported that the addition of efflux pump inhibitors wereunable to potentiate the antibiofilm activity of calcium hydroxide and nanoparticles of chitosan [133].While this lack of effectiveness of calcium hydroxide was also true for multi-species biofilms, additionchitosan nanoparticles to calcium hydroxide appears to enhance the bacterial killing in a multi-speciesmodel over a 7 and 14 day period [134]. Nevertheless, it is unknown if this strategy per se can removebiofilms from within the radicular space. Furthermore, research is lacking in terms of the ability ofintracanal medicaments to penetrate the EPS matrix of biofilms.

Antibiotic combinations have been studied over the past few years as a regimen duringregenerative endodontic strategies. The literature is inconsistent on the effectiveness of double andtriple antibiotic pastes (DAP and TAP) respectively against mono- and multi-species biofilms. It hasbeen shown that TAP is significantly better than calcium hydroxide and chlorhexidine in disruptingbiofilms of E. fecalis [6]. It has been suggested that 1 mg/mL DAP is needed to demonstrate anysignificant antibiofilm activity [135]. In addition, polymer nanofibers with TAP has been shown tobring about significant bacterial killing in a dual-species model composed of Actinomyces naeslundii andE. fecalis [136]. However, the possibility of root canal bacteria to develop resistance to antibiotics andpossible allergic reactions in patients, remain important concerns with this category of medicaments.With the current available evidence, it remains unclear if intracanal medicaments are effective againstmulti-species biofilms.

5.2. Irrigant Activation Mechanisms

Complexity of the root canal anatomy and tenacious nature of the biofilms dictate that simpledelivery of antimicrobial agents is not sufficient for disinfection of root canal systems. The focus ison developing methods that will satisfactorily deliver these antimicrobial agents into the complexanatomy, interfere with the adhesive mechanisms by inducing shear stress and disrupt the biofilms.

5.2.1. Sonics and Ultrasonics

Ultrasonic agitation can cause dis-agglomeration of the bacterial biofilm, thus re-suspendingthe bacteria in planktonic form which are then, more susceptible to antimicrobial irrigants.Also, any cavitation that may be produced, would cause temporary weakening of the cell membrane,thereby increasing the bacterial cell permeability to antimicrobial irrigants [137–139].

There is wide variability in the endodontic literature with regards to effectiveness of sonicand ultrasonics in removal of smear layer as well as antibacterial activity. In part, this may bedue to differences in the study design, with the parameters for using these activation methodsbeing inconsistent. Notable differences concerned include the volume of irrigating solution used,concentration, activation cycle and replenishment cycle of the solution, which result in inconclusiveresults [140,141]. From a logical standpoint, agitation of irrigating agents with sonic or ultrasonicshould result in shear stresses that may cause detachment of the biofilms from the root canal walls [140].

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It will also enable better penetration of irrigating agents into the lateral channels of the root canalsystem allowing better disinfection. However, there appears to be no strong evidence to demonstratethe clinical effectiveness of this approach.

5.2.2. Light: Non-Coherent (Photoactivated Disinfection) and Coherent (Laser Activated Disinfection)

Since the concept of photoactivated disinfection against root canal biofilms has been discussedalready, this section will only focus on the role of lasers against biofilms within root canals. Incompletedissolution of intracanal biofilm seems to be a common finding after chemo-mechanical preparation ofthe root canal system [142,143]. Lasers that have a wavelength interacting with water molecules havebeen used to produce cavitation in liquids. When laser irradiation pulses, the cavitation effect producesa shockwave that can move the irrigating solution within the canal. One brand of Erbium:YAG(Er:YAG) laser propose its use in combination with a special tip to achieve the so-called Photon-inducedphotoacoustic streaming (PIPS) or irrigant in the canal. This device has been researched for removingdebris and smear layer from the root canal system and the results seem positive [144].

There are only a few studies that evaluated laser activation of irrigants using a bioflm model;one of them examined the cleaning of biofilm-infected dentin on a bovine root canal comparing itwith sonic or ultrasonic activation and needle irrigation. The authors showed favorable results forPIPS when compared to the other irrigant agitation methods [77]. Neelakantan et al., demonstratedthat both diode and Er:YAG lasers were more effective than ultrasonic activation or syringe irrigationmethod for removing E. fecalis biofilms. However, this study reported no significant difference betweenEr:YAG and diode laser when a new irrigating agent (sodium hypochlorite mixed with etidronic acid)was used [81].

5.2.3. Microbubble Emulsion

Halford et al., were the first to employ a microbubble emulsion to enhance the effect of sonicand ultrasonic agitation of sodium hypochlorite [145]. Essentially, the technique employs unstablegas-filled microbubbles that expand when exposed to ultrasonic waves. The dynamics thereby inducedin the fluid would help in detaching surface adherent bacteria or biofilm destruction. In addition, itmay also generate reactive oxygen species to exhibit an antibacterial effect. Microbubble emulsionin combination with ultrasonic agitation was shown to be superior than with sonic agitation [145].This approach appears clinically interesting and warrants further research.

6. Future Directions

Despite the increasing knowledge of the microbial status of root canal systems, much stillremains unknown. The reported success rates of root canal treatment have not undergonesignificant improvement [146–149]. From the clinical perspective, it is important to understand theaetiopathogenesis of periradicular periodontitis as a disease caused by microbial infection of the rootcanal system. Even though we know that root canal biofilms are complex, the literature unfortunatelydoes not seem to offer due credence to understanding the dynamics between the components of abiofilm. Crosstalk between bacteria is a paradigm that has not be sufficiently studied thus far in thecontext of endodontic disease.

The authors of this paper call into action, the need for better understanding of the interactionsbetween microbes in biofilms and how each organism influences the other. This, coupled with targetedtherapeutic strategies may help improve the success rates of root canal treatment. Such strategiesmust focus on a step-wise approach from mono- to multispecies biofilms so as to develop a sufficientknowledge base on their mechanisms at a cellular level.

7. Conclusions

The root canal biofilm is a very complex, organized entity and it is difficult, but not impossible toduplicate its characteristics in in vitro experiments. Within root canal systems, the complexity is not

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only related to the nature of the biofilm, but also the complex anatomy, which houses tissue along withbiofilms and removal of such biomasses is as relevant as being able to kill bacteria in biofilms.

Studies on monospecies or dual species biofilms may over-simplify this ecological phenomenonand may not be a true reflection of the results achievable in the clinical scenario.

The authors recommend that future studies should offer due credence to the complexity of themicrobial biofilm and evaluate models to re-evaluate removal of the biomass from the root canals inaddition to evaluating the action of novel antimicrobial agents on complex biofilms.

Acknowledgments: This work was supported by The University of Hong Kong Seed Fund for Basic Research(Grant number 201702159011).

Author Contributions: Conception and design: Prasanna Neelakantan and Gary Shun Pan Cheung; Literaturereview: Monica Romero, Jorge Vera, Prasanna Neelakantan, Umer Daood; Critical revision for importantintellectual content: Gary Shun Pan Cheung, Asad U. Khan, Aixin Yan; Final approval: Prasanna Neelakantan,Monica Romero, Jorge Vera, Umer Daood, Asad U. Khan, Aixin Yan, Gary Shun Pan Cheung.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Flemming, H.C.; Wingender, J.; Szewzyk, U.; Steinberg, P.; Rice, S.A.; Kjelleberg, S. Biofilms: An emergentform of bacterial life. Nat. Rev. Microbiol. 2016, 14, 563–575. [CrossRef] [PubMed]

2. Marsh, P.D. Dental plaque as a microbial biofilm. Caries Res. 2004, 38, 204–211. [CrossRef] [PubMed]3. Costerton, J.W.; Stewart, P.S. Battling biofilms. Sci. Am. 2001, 285, 74–81. [CrossRef] [PubMed]4. Diaz, P.I. Microbial diversity and interactions in subgingival biofilm communities. Front. Oral Biol. 2012, 15,

17–40. [PubMed]5. Caggianiello, G.; Kleerebezem, M.; Spano, G. Exopolysaccharides produced by lactic acid bacteria: From

health-promoting benefits to stress tolerance mechanisms. Appl. Microbiol. Biotechnol. 2016, 100, 3877–3886.[CrossRef] [PubMed]

6. Devaraj, S.; Jagannathan, N.; Neelakantan, P. Antibiofilm efficacy of photoactivated curcumin, triple anddouble antibiotic paste, 2% chlorhexidine and calcium hydroxide against Enterococcus fecalis in vitro.Sci. Rep. 2016, 6, 24797. [CrossRef] [PubMed]

7. Patel, R. Biofilms and antimicrobial resistance. Clin. Orthop. Relat. Res. 2005, 437, 41–47. [CrossRef]8. Dunavant, T.R.; Regan, J.D.; Glickman, G.N.; Solomon, E.S.; Honeyman, A.L. Comparative evaluation of

endodontic irrigants against Enterococcus faecalis biofilms. J. Endod. 2006, 32, 527–531. [CrossRef] [PubMed]9. Siqueira, J.F., Jr.; Rocas, I.N. Optimising single-visit disinfection with supplementary approaches: A quest

for predictability. Aust. Endod. J. 2011, 37, 92–98. [CrossRef] [PubMed]10. Zehnder, M. Root canal irrigants. J. Endod. 2006, 32, 389–398. [CrossRef] [PubMed]11. Haapasalo, M.; Udnæs, T.; Endal, U. Persistent, recurrent, and acquired infection of the root canal system

post-treatment. Endod. Top. 2003, 6, 29–56. [CrossRef]12. Ricucci, D.; Siqueira, J.F., Jr. Biofilms and apical periodontitis: Study of prevalence and association with

clinical and histopathologic findings. J. Endod. 2010, 36, 1277–1288. [CrossRef] [PubMed]13. Alves, F.R.; Almeida, B.M.; Neves, M.A.; Moreno, J.O.; Rocas, I.N.; Siqueira, J.F., Jr. Disinfecting oval-shaped

root canals: Effectiveness of different supplementary approaches. J. Endod. 2011, 37, 496–501. [CrossRef][PubMed]

14. Susin, L.; Liu, Y.; Yoon, J.C.; Parente, J.M.; Loushine, R.J.; Ricucci, D.; Bryan, T.; Weller, R.N.; Pashley, D.H.;Tay, F.R. Canal and isthmus debridement efficacies of two irrigant agitation techniques in a closed system.Int. Endod. J. 2010, 43, 1077–1090. [CrossRef] [PubMed]

15. Antunes, H.S.; Rocas, I.N.; Alves, F.R.; Siqueira, J.F., Jr. Total and Specific Bacterial Levels in the Apical RootCanal System of Teeth with Post-treatment Apical Periodontitis. J. Endod. 2015, 41, 1037–1042. [CrossRef][PubMed]

16. Winkler, K.C.; van Amerongen, J. Bacteriologic results from 4000 root canal cultures. Oral Surg. Oral Med.Oral Pathol. 1959, 12, 857–875. [CrossRef]

17. Bystrom, A.; Sundqvist, G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentationin endodontic therapy. Scand. J. Dent. Res. 1981, 89, 321–328. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 15 of 21

18. Sundqvist, G.K.; Eckerbom, M.I.; Larsson, A.P.; Sjogren, U.T. Capacity of anaerobic bacteria from necroticdental pulps to induce purulent infections. Infect. Immun. 1979, 25, 685–693. [PubMed]

19. Provenzano, J.C.; Siqueira, J.F., Jr.; Rocas, I.N.; Domingues, R.R.; Paes Leme, A.F.; Silva, M.R. Metaproteomeanalysis of endodontic infections in association with different clinical conditions. PLoS ONE 2013, 8, e76108.[CrossRef] [PubMed]

20. Rocas, I.N.; Siqueira, J.F., Jr. Identification of bacteria enduring endodontic treatment procedures bya combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach.J. Endod. 2010, 36, 45–52. [CrossRef] [PubMed]

21. Alves, F.R.; Andrade-Junior, C.V.; Marceliano-Alves, M.F.; Perez, A.R.; Rocas, I.N.; Versiani, M.A.;Sousa-Neto, M.D.; Provenzano, J.C.; Siqueira, J.F., Jr. Adjunctive Steps for Disinfection of theMandibular Molar Root Canal System: A Correlative Bacteriologic, Micro-Computed Tomography, andCryopulverization Approach. J. Endod. 2016, 42, 1667–1672. [CrossRef] [PubMed]

22. Anderson, A.C.; Hellwig, E.; Vespermann, R.; Wittmer, A.; Schmid, M.; Karygianni, L.; Al-Ahmad, A.Comprehensive analysis of secondary dental root canal infections: A combination of culture andculture-independent approaches reveals new insights. PLoS ONE 2012, 7, e49576. [CrossRef] [PubMed]

23. Lins, R.X.; de Oliveira Andrade, A.; Hirata Junior, R.; Wilson, M.J.; Lewis, M.A.; Williams, D.W.; Fidel, R.A.Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections.J. Dent. 2013, 41, 779–786. [CrossRef] [PubMed]

24. Ran, S.; He, Z.; Liang, J. Survival of Enterococcus faecalis during alkaline stress: Changes in morphology,ultrastructure, physiochemical properties of the cell wall and specific gene transcripts. Arch. Oral Biol. 2013,58, 1667–1676. [CrossRef] [PubMed]

25. Love, R.M. Enterococcus faecalis—A mechanism for its role in endodontic failure. Int. Endod. J. 2001, 34,399–405. [CrossRef] [PubMed]

26. Wang, J.; Jiang, Y.; Chen, W.; Zhu, C.; Liang, J. Bacterial flora and extraradicular biofilm associated withthe apical segment of teeth with post-treatment apical periodontitis. J. Endod. 2012, 38, 954–959. [CrossRef][PubMed]

27. Guo, H.; Gao, C.; Zhang, C.; Zheng, S.; Yue, L. Morphology of bacterial flora in root canals associated withapical abscesses. Chin. Med. J. 2014, 127, 3254–3258. [PubMed]

28. Siqueira, J.F., Jr. Microbial causes of endodontic flare-ups. Int. Endod. J. 2003, 36, 453–463. [CrossRef][PubMed]

29. Lin, J.; Shen, Y.; Haapasalo, M. A comparative study of biofilm removal with hand, rotary nickel-titanium,and self-adjusting file instrumentation using a novel in vitro biofilm model. J. Endod. 2013, 39, 658–663.[CrossRef] [PubMed]

30. Schroeder, M.; Brooks, B.D.; Brooks, A.E. The Complex Relationship between Virulence and AntibioticResistance. Genes 2017, 8, 39. [CrossRef] [PubMed]

31. Zehnder, M.; Belibasakis, G.N. On the dynamics of root canal infections-what we understand and what wedon’t. Virulence 2015, 6, 216–222. [CrossRef] [PubMed]

32. Davey, M.E.; O’Toole, G.A. Microbial biofilms: From ecology to molecular genetics. Microbiol. Mol. Biol. Rev.2000, 64, 847–867. [CrossRef] [PubMed]

33. Flemming, H.C. EPS-Then and Now. Microorganisms 2016, 4, 41. [CrossRef] [PubMed]34. Barbosa-Ribeiro, M.; De-Jesus-Soares, A.; Zaia, A.A.; Ferraz, C.C.; Almeida, J.F.; Gomes, B.P. Antimicrobial

Susceptibility and Characterization of Virulence Genes of Enterococcus faecalis Isolates from Teeth withFailure of the Endodontic Treatment. J. Endod. 2016, 42, 1022–1028. [CrossRef] [PubMed]

35. Portenier, I.; Waltimo, T.; Orstavik, D.; Haapasalo, M. The susceptibility of starved, stationary phase, andgrowing cells of Enterococcus faecalis to endodontic medicaments. J. Endod. 2005, 31, 380–386. [CrossRef][PubMed]

36. Gerdes, K.; Semsey, S. Microbiology: Pumping persisters. Nature 2016, 534, 41–42. [CrossRef] [PubMed]37. Kaldalu, N.; Hauryliuk, V.; Tenson, T. Persisters-as elusive as ever. Appl. Microbiol. Biotechnol. 2016, 100,

6545–6553. [CrossRef] [PubMed]38. Zhao, J.; Shen, Y.; Haapasalo, M.; Wang, Z.; Wang, Q. A 3D numerical study of antimicrobial persistence in

heterogeneous multi-species biofilms. J. Theor. Biol. 2016, 392, 83–98. [CrossRef] [PubMed]39. Majumdar, S.; Pal, S. Cross-species communication in bacterial world. J. Cell Commun. Signal. 2017, 11,

187–190. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 16 of 21

40. Mangwani, N.; Kumari, S.; Das, S. Bacterial biofilms and quorum sensing: Fidelity in bioremediationtechnology. Biotechnol. Genet. Eng. Rev. 2016, 32, 43–73. [CrossRef] [PubMed]

41. Lillicrap, A.; Macken, A.; Wennberg, A.C.; Grung, M.; Rundberget, J.T.; Fredriksen, L.; Scheie, A.A.;Benneche, T.; d’Auriac, M.A. Environmental fate and effects of novel quorum sensing inhibitors thatcan control biofilm formation. Chemosphere 2016, 164, 52–58. [CrossRef] [PubMed]

42. Chavez de Paz, L.E.; Davies, J.R.; Bergenholtz, G.; Svensater, G. Strains of Enterococcus faecalis differ intheir ability to coexist in biofilms with other root canal bacteria. Int. Endod. J. 2015, 48, 916–925. [CrossRef][PubMed]

43. He, Z.; Liang, J.; Zhou, W.; Xie, Q.; Tang, Z.; Ma, R.; Huang, Z. Effect of the quorum-sensing luxS gene onbiofilm formation by Enterococcus faecalis. Eur. J. Oral Sci. 2016, 124, 234–240. [CrossRef] [PubMed]

44. Gabrilska, R.A.; Rumbaugh, K.P. Biofilm models of polymicrobial infection. Future Microbiol. 2015, 10,1997–2015. [CrossRef] [PubMed]

45. Ruiz-Linares, M.; Aguado-Perez, B.; Baca, P.; Arias-Moliz, M.T.; Ferrer-Luque, C.M. Efficacy of antimicrobialsolutions against polymicrobial root canal biofilm. Int. Endod. J. 2017, 50, 77–83. [CrossRef] [PubMed]

46. Chavez de Paz, L.E. Development of a multispecies biofilm community by four root canal bacteria. J. Endod.2012, 38, 318–323. [CrossRef] [PubMed]

47. Gilbert, P.; Maira-Litran, T.; McBain, A.J.; Rickard, A.H.; Whyte, F.W. The physiology and collectiverecalcitrance of microbial biofilm communities. Adv. Microb. Physiol. 2002, 46, 202–256. [PubMed]

48. Wuertz, S.; Okabe, S.; Hausner, M. Microbial communities and their interactions in biofilm systems:An overview. Water Sci. Technol. 2004, 49, 327–336. [PubMed]

49. Chavez de Paz, L.E. Image analysis software based on color segmentation for characterization of viabilityand physiological activity of biofilms. Appl. Environ. Microbiol. 2009, 75, 1734–1739. [CrossRef] [PubMed]

50. Lebeaux, D.; Chauhan, A.; Rendueles, O.; Beloin, C. From in vitro to in vivo models of bacterialbiofilm-related infections. Pathogens 2013, 2, 288–356. [CrossRef] [PubMed]

51. Peters, B.M.; Jabra-Rizk, M.A.; O’May, G.A.; Costerton, J.W.; Shirtliff, M.E. Polymicrobial interactions: Impacton pathogenesis and human disease. Clin. Microbiol. Rev. 2012, 25, 193–213. [CrossRef] [PubMed]

52. McLean, J.S. Advancements toward a systems level understanding of the human oral microbiome.Front. Cell. Infect. Microbiol. 2014, 4, 98. [CrossRef] [PubMed]

53. Qayyum, S.; Sharma, D.; Bisht, D.; Khan, A.U. Protein translation machinery holds a key fortransition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach.Biochem. Biophys. Res. Commun. 2016, 474, 652–659. [CrossRef] [PubMed]

54. Farmer, J.T.; Shimkevitch, A.V.; Reilly, P.S.; Mlynek, K.D.; Jensen, K.S.; Callahan, M.T.; Bushaw-Newton, K.L.;Kaplan, J.B. Environmental bacteria produce abundant and diverse antibiofilm compounds. J. Appl. Microbiol.2014, 117, 1663–1673. [CrossRef] [PubMed]

55. Macia, M.D.; Rojo-Molinero, E.; Oliver, A. Antimicrobial susceptibility testing in biofilm-growing bacteria.Clin. Microbiol. Infect. 2014, 20, 981–990. [CrossRef] [PubMed]

56. Peeters, E.; Nelis, H.J.; Coenye, T. Comparison of multiple methods for quantification of microbial biofilmsgrown in microtiter plates. J. Microbiol. Methods 2008, 72, 157–165. [CrossRef] [PubMed]

57. Busscher, H.J.; van der Mei, H.C. Microbial adhesion in flow displacement systems. Clin. Microbiol. Rev.2006, 19, 127–141. [CrossRef] [PubMed]

58. Rickard, A.H.; McBain, A.J.; Stead, A.T.; Gilbert, P. Shear rate moderates community diversity in freshwaterbiofilms. Appl. Environ. Microbiol. 2004, 70, 7426–7435. [CrossRef] [PubMed]

59. Coenye, T.; de Prijck, K.; de Wever, B.; Nelis, H.J. Use of the modified Robbins device to study the in vitrobiofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medicaldevices. J. Appl. Microbiol. 2008, 105, 733–740. [CrossRef] [PubMed]

60. Honraet, K.; Nelis, H.J. Use of the modified robbins device and fluorescent staining to screen plant extractsfor the inhibition of S. mutans biofilm formation. J. Microbiol. Methods 2006, 64, 217–224. [CrossRef] [PubMed]

61. Shin, S.; Ahmed, I.; Hwang, J.; Seo, Y.; Lee, E.; Choi, J.; Moon, S.; Hong, J.W. A Microfluidic Approach toInvestigating a Synergistic Effect of Tobramycin and Sodium Dodecyl Sulfate on Pseudomonas aeruginosaBiofilms. Anal. Sci. 2016, 32, 67–73. [CrossRef] [PubMed]

62. Khandurina, J.; Guttman, A. Bioanalysis in microfluidic devices. J. Chromatogr. A 2002, 943, 159–183.[CrossRef]

Int. J. Mol. Sci. 2017, 18, 1748 17 of 21

63. Lion, N.; Rohner, T.C.; Dayon, L.; Arnaud, I.L.; Damoc, E.; Youhnovski, N.; Wu, Z.Y.; Roussel, C.; Josserand, J.;Jensen, H.; et al. Microfluidic systems in proteomics. Electrophoresis 2003, 24, 3533–3562. [CrossRef] [PubMed]

64. Yawata, Y.; Toda, K.; Setoyama, E.; Fukuda, J.; Suzuki, H.; Uchiyama, H.; Nomura, N. Monitoring biofilmdevelopment in a microfluidic device using modified confocal reflection microscopy. J. Biosci. Bioeng. 2010,110, 377–380. [CrossRef] [PubMed]

65. Peterson, B.W.; He, Y.; Ren, Y.; Zerdoum, A.; Libera, M.R.; Sharma, P.K.; van Winkelhoff, A.J.; Neut, D.;Stoodley, P.; van der Mei, H.C.; et al. Viscoelasticity of biofilms and their recalcitrance to mechanical andchemical challenges. FEMS Microbiol. Rev. 2015, 39, 234–245. [CrossRef] [PubMed]

66. Ma, J.; Wang, Z.; Shen, Y.; Haapasalo, M. A new noninvasive model to study the effectiveness of dentindisinfection by using confocal laser scanning microscopy. J. Endod. 2011, 37, 1380–1385. [CrossRef] [PubMed]

67. Zapata, R.O.; Bramante, C.M.; de Moraes, I.G.; Bernardineli, N.; Gasparoto, T.H.; Graeff, M.S.;Campanelli, A.P.; Garcia, R.B. Confocal laser scanning microscopy is appropriate to detect viability ofEnterococcus faecalis in infected dentin. J. Endod. 2008, 34, 1198–1201. [CrossRef] [PubMed]

68. Filoche, S.K.; Coleman, M.J.; Angker, L.; Sissons, C.H. A fluorescence assay to determine the viable biomassof microcosm dental plaque biofilms. J. Microbiol. Methods 2007, 69, 489–496. [CrossRef] [PubMed]

69. Alhede, M.; Qvortrup, K.; Liebrechts, R.; Hoiby, N.; Givskov, M.; Bjarnsholt, T. Combination of microscopictechniques reveals a comprehensive visual impression of biofilm structure and composition. FEMS Immun.Med. Microbiol. 2012, 65, 335–342. [CrossRef] [PubMed]

70. Kelleher, S.M.; Habimana, O.; Lawler, J.; O’Reilly, B.; Daniels, S.; Casey, E.; Cowley, A. Cicada WingSurface Topography: An Investigation into the Bactericidal Properties of Nanostructural Features.ACS Appl. Mater. Interfaces 2016, 8, 14966–14974. [CrossRef] [PubMed]

71. Stewart, P.S.; Costerton, J.W. Antibiotic resistance of bacteria in biofilms. Lancet 2001, 358, 135–138. [CrossRef]72. Good, M.; El, K.I.; Hussey, D.L. Endodontic ‘solutions’ part 1: A literature review on the use of endodontic

lubricants, irrigants and medicaments. Dent. Update 2012, 39, 239–246. [CrossRef] [PubMed]73. Haapasalo, M.; Shen, Y.; Wang, Z.; Gao, Y. Irrigation in endodontics. Br. Dent. J. 2014, 216, 299–303.

[CrossRef] [PubMed]74. Arias-Moliz, M.T.; Ferrer-Luque, C.M.; Espigares-Garcia, M.; Baca, P. Enterococcus faecalis biofilms eradication

by root canal irrigants. J. Endod. 2009, 35, 711–714. [CrossRef] [PubMed]75. Wong, D.T.; Cheung, G.S. Extension of bactericidal effect of sodium hypochlorite into dentinal tubules.

J. Endod. 2014, 40, 825–829. [CrossRef] [PubMed]76. Yang, Y.; Shen, Y.; Wang, Z.; Huang, X.; Maezono, H.; Ma, J.; Cao, Y.; Haapasalo, M. Evaluation of the

Susceptibility of Multispecies Biofilms in Dentinal Tubules to Disinfecting Solutions. J. Endod. 2016, 42,1246–1250. [CrossRef] [PubMed]

77. Ordinola-Zapata, R.; Bramante, C.M.; Aprecio, R.M.; Handysides, R.; Jaramillo, D.E. Biofilm removal by 6%sodium hypochlorite activated by different irrigation techniques. Int. Endod. J. 2014, 47, 659–666. [CrossRef][PubMed]

78. Tartari, T.; Bachmann, L.; Maliza, A.G.; Andrade, F.B.; Duarte, M.A.; Bramante, C.M. Tissue dissolution andmodifications in dentin composition by different sodium hypochlorite concentrations. J. Appl. Oral Sci. 2016,24, 291–298. [CrossRef] [PubMed]

79. Ozdemir, H.O.; Buzoglu, H.D.; Calt, S.; Stabholz, A.; Steinberg, D. Effect of ethylenediaminetetraacetic acidand sodium hypochlorite irrigation on Enterococcus faecalis biofilm colonization in young and old humanroot canal dentin: In vitro study. J. Endod. 2010, 36, 842–846. [CrossRef] [PubMed]

80. Jungbluth, H.; Marending, M.; De-Deus, G.; Sener, B.; Zehnder, M. Stabilizing sodium hypochlorite at highpH: Effects on soft tissue and dentin. J. Endod. 2011, 37, 693–696. [CrossRef] [PubMed]

81. Neelakantan, P.; Cheng, C.Q.; Mohanraj, R.; Sriraman, P.; Subbarao, C.; Sharma, S. Antibiofilm activity ofthree irrigation protocols activated by ultrasonic, diode laser or Er:YAG laser in vitro. Int. Endod. J. 2015, 48,602–610. [CrossRef] [PubMed]

82. De Hemptinne, F.; Slaus, G.; Vandendael, M.; Jacquet, W.; de Moor, R.J.; Bottenberg, P. In Vivo IntracanalTemperature Evolution during Endodontic Treatment after the Injection of Room Temperature or PreheatedSodium Hypochlorite. J. Endod. 2015, 41, 1112–1115. [CrossRef] [PubMed]

83. Neelakantan, P.; Devaraj, S.; Jagannathan, N. Histologic Assessment of Debridement of the Root CanalIsthmus of Mandibular Molars by Irrigant Activation Techniques Ex Vivo. J. Endod. 2016, 42, 1268–1272.[CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 18 of 21

84. Rosen, E.; Tsesis, I.; Elbahary, S.; Storzi, N.; Kolodkin-Gal, I. Eradication of Enterococcus faecalis Biofilms onHuman Dentin. Front. Microbiol. 2016, 7, 2055. [CrossRef] [PubMed]

85. Bernardi, A.; Teixeira, C.S. The properties of chlorhexidine and undesired effects of its use in endodontics.Quintessence Int. 2015, 46, 575–582. [PubMed]

86. Ercan, E.; Ozekinci, T.; Atakul, F.; Gul, K. Antibacterial activity of 2% chlorhexidine gluconate and 5.25%sodium hypochlorite in infected root canal: In vivo study. J. Endod. 2004, 30, 84–87. [CrossRef] [PubMed]

87. Arias-Moliz, M.T.; Ferrer-Luque, C.M.; Gonzalez-Rodriguez, M.P.; Valderrama, M.J.; Baca, P. Eradicationof Enterococcus faecalis biofilms by cetrimide and chlorhexidine. J. Endod. 2010, 36, 87–90. [CrossRef][PubMed]

88. Baca, P.; Mendoza-Llamas, M.L.; Arias-Moliz, M.T.; Gonzalez-Rodriguez, M.P.; Ferrer-Luque, C.M. Residualeffectiveness of final irrigation regimens on Enteroccus faecalis-infected root canals. J. Endod. 2011, 37,1121–1123. [CrossRef] [PubMed]

89. Baca, P.; Junco, P.; Arias-Moliz, M.T.; Castillo, F.; Rodriguez-Archilla, A.; Ferrer-Luque, C.M. Antimicrobialsubstantivity over time of chlorhexidine and cetrimide. J. Endod. 2012, 38, 927–930. [CrossRef] [PubMed]

90. Shen, Y.; Stojicic, S.; Qian, W.; Olsen, I.; Haapasalo, M. The synergistic antimicrobial effect by mechanicalagitation and two chlorhexidine preparations on biofilm bacteria. J. Endod. 2010, 36, 100–104. [CrossRef][PubMed]

91. Arias-Moliz, M.T.; Ordinola-Zapata, R.; Baca, P.; Ruiz-Linares, M.; Garcia Garcia, E.; Hungaro Duarte, M.A.;Monteiro Bramante, C.; Ferrer-Luque, C.M. Antimicrobial activity of Chlorhexidine, Peracetic acid andSodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms. Int. Endod. J. 2015,48, 1188–1193. [CrossRef] [PubMed]

92. Bukhary, S.; Balto, H. Antibacterial Efficacy of Octenisept, Alexidine, Chlorhexidine, and SodiumHypochlorite against Enterococcus faecalis Biofilms. J. Endod. 2017, 43, 643–647. [CrossRef] [PubMed]

93. Zorko, M.; Jerala, R. Alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid andprevent cell activation by antibiotics. J. Antimicrob. Chemother. 2008, 62, 730–737. [CrossRef] [PubMed]

94. Kim, H.S.; Woo Chang, S.; Baek, S.H.; Han, S.H.; Lee, Y.; Zhu, Q.; Kum, K.Y. Antimicrobial effect of alexidineand chlorhexidine against Enterococcus faecalis infection. Int. J. Oral Sci. 2013, 5, 26–31. [CrossRef] [PubMed]

95. Silveira, L.F.; Baca, P.; Arias-Moliz, M.T.; Rodriguez-Archilla, A.; Ferrer-Luque, C.M. Antimicrobial activityof alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm. Int. J. Oral Sci.2013, 5, 146–149. [CrossRef] [PubMed]

96. Tirali, R.E.; Bodur, H.; Sipahi, B.; Sungurtekin, E. Evaluation of the antimicrobial activities of chlorhexidinegluconate, sodium hypochlorite and octenidine hydrochloride in vitro. Aust. Endod. J. 2013, 39, 15–18.[CrossRef] [PubMed]

97. Eldeniz, A.U.; Guneser, M.B.; Akbulut, M.B. Comparative antifungal efficacy of light-activated disinfectionand octenidine hydrochloride with contemporary endodontic irrigants. Lasers Med. Sci. 2015, 30, 669–675.[CrossRef] [PubMed]

98. Tello-Barbaran, J.; Nakata, H.M.; Salcedo-Moncada, D.; Bramante, C.M.; Ordinola-Zapata, R. Theantimicrobial effect of iodine-potassium iodide after cleaning and shaping procedures in mesial root canalsof mandibular molars. Acta Odontol. Latinoam. 2010, 23, 244–247. [PubMed]

99. Wang, Z.; Shen, Y.; Ma, J.; Haapasalo, M. The effect of detergents on the antibacterial activity of disinfectingsolutions in dentin. J. Endod. 2012, 38, 948–953. [CrossRef] [PubMed]

100. Soares, J.A.; Roque de Carvalho, M.A.; Cunha Santos, S.M.; Mendonca, R.M.; Ribeiro-Sobrinho, A.P.;Brito-Junior, M.; Magalhaes, P.P.; Santos, M.H.; de Macedo Farias, L. Effectiveness of chemomechanicalpreparation with alternating use of sodium hypochlorite and EDTA in eliminating intracanal Enterococcusfaecalis biofilm. J. Endod. 2010, 36, 894–898. [CrossRef] [PubMed]

101. Mohammadi, Z.; Shalavi, S. Antifungal effects of root canal irrigants and medicaments. An update review.N. Y. State Dent. J. 2014, 80, 58–63. [PubMed]

102. Ferrer-Luque, C.M.; Arias-Moliz, M.T.; Gonzalez-Rodriguez, M.P.; Baca, P. Antimicrobial activity of maleicacid and combinations of cetrimide with chelating agents against Enterococcus faecalis biofilm. J. Endod.2010, 36, 1673–1675. [CrossRef] [PubMed]

103. Ordinola-Zapata, R.; Bramante, C.M.; Garcia, R.B.; de Andrade, F.B.; Bernardineli, N.; de Moraes, I.G.;Duarte, M.A. The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infecteddentin. Acta Odontol. Scand. 2013, 71, 424–431. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 19 of 21

104. Lottanti, S.; Gautschi, H.; Sener, B.; Zehnder, M. Effects of ethylenediaminetetraacetic, etidronic and peraceticacid irrigation on human root dentine and the smear layer. Int. Endod. J. 2009, 42, 335–343. [CrossRef][PubMed]

105. Prabhakar, J.; Senthilkumar, M.; Priya, M.S.; Mahalakshmi, K.; Sehgal, P.K.; Sukumaran, V.G. Evaluation ofantimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodiumhypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: An in vitro study. J. Endod.2010, 36, 83–86. [CrossRef] [PubMed]

106. Clegg, M.S.; Vertucci, F.J.; Walker, C.; Belanger, M.; Britto, L.R. The effect of exposure to irrigant solutions onapical dentin biofilms in vitro. J. Endod. 2006, 32, 434–437. [CrossRef] [PubMed]

107. Stojicic, S.; Shen, Y.; Qian, W.; Johnson, B.; Haapasalo, M. Antibacterial and smear layer removal ability ofa novel irrigant, QMiX. Int. Endod. J. 2012, 45, 363–371. [CrossRef] [PubMed]

108. Wang, Z.; Shen, Y.; Haapasalo, M. Effectiveness of endodontic disinfecting solutions against young and oldEnterococcus faecalis biofilms in dentin canals. J. Endod. 2012, 38, 1376–1379. [CrossRef] [PubMed]

109. Xie, Q.; Johnson, B.R.; Wenckus, C.S.; Fayad, M.I.; Wu, C.D. Efficacy of berberine, an antimicrobial plantalkaloid, as an endodontic irrigant against a mixed-culture biofilm in an in vitro tooth model. J. Endod. 2012,38, 1114–1147. [CrossRef] [PubMed]

110. Wei, G.X.; Xu, X.; Wu, C.D. In vitro synergism between berberine and miconazole against planktonic andbiofilm Candida cultures. Arch. Oral Biol. 2011, 56, 565–572. [CrossRef] [PubMed]

111. Neelakantan, P.; Cheng, C.Q.; Ravichandran, V.; Mao, T.; Sriraman, P.; Sridharan, S.; Subbarao, C.; Sharma, S.;Kishen, A. Photoactivation of curcumin and sodium hypochlorite to enhance antibiofilm efficacy in rootcanal dentin. Photodiagnosis Photodyn. Ther. 2015, 12, 108–114. [CrossRef] [PubMed]

112. Kishen, A.; Shi, Z.; Shrestha, A.; Neoh, K.G. An investigation on the antibacterial and antibiofilm efficacy ofcationic nanoparticulates for root canal disinfection. J. Endod. 2008, 34, 1515–1520. [CrossRef] [PubMed]

113. Shrestha, A.; Kishen, A. Antibacterial Nanoparticles in Endodontics: A Review. J. Endod. 2016, 42, 1417–1426.[CrossRef] [PubMed]

114. Wu, D.; Fan, W.; Kishen, A.; Gutmann, J.L.; Fan, B. Evaluation of the antibacterial efficacy of silvernanoparticles against Enterococcus faecalis biofilm. J. Endod. 2014, 40, 285–290. [CrossRef] [PubMed]

115. Shrestha, A.; Hamblin, M.R.; Kishen, A. Photoactivated rose bengal functionalized chitosan nanoparticlesproduce antibacterial/biofilm activity and stabilize dentin-collagen. Nanomed. Nanotechnol. Biol. Med. 2014,10, 491–501. [CrossRef] [PubMed]

116. Ferrari, M.; Mason, P.N.; Goracci, C.; Pashley, D.H.; Tay, F.R. Collagen degradation in endodontically treatedteeth after clinical function. J. Dent. Res. 2004, 83, 414–419. [CrossRef] [PubMed]

117. Shrestha, A.; Hamblin, M.R.; Kishen, A. Characterization of a conjugate between Rose Bengal andchitosan for targeted antibiofilm and tissue stabilization effects as a potential treatment of infected dentin.Antimicrob. Agents Chemother. 2012, 56, 4876–4884. [CrossRef] [PubMed]

118. Persadmehr, A.; Torneck, C.D.; Cvitkovitch, D.G.; Pinto, V.; Talior, I.; Kazembe, M.; Shrestha, S.;McCulloch, C.A.; Kishen, A. Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagendegradation in vitro. J. Endod. 2014, 40, 703–709. [CrossRef] [PubMed]

119. Li, S.; Cui, S.; Yin, D.; Zhu, Q.; Ma, Y.; Qian, Z.; Gu, Y. Dual antibacterial activities of a chitosan-modifiedupconversion photodynamic therapy system against drug-resistant bacteria in deep tissue. Nanoscale 2017, 9,3912–3924. [CrossRef] [PubMed]

120. Moczek, L.; Nowakowska, M. Novel water-soluble photosensitizers from chitosan. Biomacromolecules 2007, 8,433–438. [CrossRef] [PubMed]

121. Severyukhina, A.N.; Petrova, N.V.; Yashchenok, A.M.; Bratashov, D.N.; Smuda, K.; Mamonova, I.A.;Yurasov, N.A.; Puchinyan, D.M.; Georgieva, R.; Baumler, H.; et al. Light-induced antibacterial activity ofelectrospun chitosan-based material containing photosensitizer. Mater. Sci. Eng. C Mater. Biol. Appl. 2017, 70,311–316. [CrossRef] [PubMed]

122. Fan, W.; Wu, D.; Ma, T.; Fan, B. Ag-loaded mesoporous bioactive glasses against Enterococcus faecalisbiofilm in root canal of human teeth. Dent. Mater. J. 2015, 34, 54–60. [CrossRef] [PubMed]

123. Veerapandian, M.; Yun, K. Functionalization of biomolecules on nanoparticles: Specialized for antibacterialapplications. Appl. Microbiol. Biotechnol. 2011, 90, 1655–1667. [CrossRef] [PubMed]

124. Shrestha, A.; Kishen, A. Antibacterial efficacy of photosensitizer functionalized biopolymeric nanoparticlesin the presence of tissue inhibitors in root canal. J. Endod. 2014, 40, 566–570. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 20 of 21

125. Ruenraroengsak, P.; Cook, J.M.; Florence, A.T. Nanosystem drug targeting: Facing up to complex realities.J. Control. Release 2010, 141, 265–276. [CrossRef] [PubMed]

126. Niazi, S.A.; Clark, D.; Do, T.; Gilbert, S.C.; Foschi, F.; Mannocci, F.; Beighton, D. The effectiveness of enzymicirrigation in removing a nutrient-stressed endodontic multispecies biofilm. Int. Endod. J. 2014, 47, 756–768.[CrossRef] [PubMed]

127. Manfredi, M.; Figini, L.; Gagliani, M.; Lodi, G. Single versus multiple visits for endodontic treatment ofpermanent teeth. Cochrane Database Syst. Rev. 2016, 12, Cd005296. [PubMed]

128. Almeida, D.O.; Chaves, S.C.; Souza, R.A.; Soares, F.F. Outcome of Single- vs. Multiple-visit EndodonticTherapy of Nonvital Teeth: A Meta-analysis. J. Contemp. Dent. Pract. 2017, 18, 330–336. [CrossRef] [PubMed]

129. De-Deus, G.; Canabarro, A. Strength of recommendation for single-visit root canal treatment: Grading thebody of the evidence using a patient-centred approach. Int. Endod. J. 2017, 50, 251–259. [CrossRef] [PubMed]

130. Kawashima, N.; Wadachi, R.; Suda, H.; Yeng, T.; Parashos, P. Root canal medicaments. Int. Dent. J. 2009, 59,5–11. [PubMed]

131. Mohammadi, Z. Antibiotics as intracanal medicaments: A review. J. Calif. Dent. Assoc. 2009, 37, 98–108.[PubMed]

132. Estrela, C.; Sydney, G.B.; Figueiredo, J.A.; Estrela, C.R. Antibacterial efficacy of intracanal medicaments onbacterial biofilm: A critical review. J. Appl. Oral Sci. 2009, 17, 1–7. [CrossRef] [PubMed]

133. Upadya, M.; Shrestha, A.; Kishen, A. Role of efflux pump inhibitors on the antibiofilm efficacy of calciumhydroxide, chitosan nanoparticles, and light-activated disinfection. J. Endod. 2011, 37, 1422–1426. [CrossRef][PubMed]

134. Del Carpio-Perochena, A.; Kishen, A.; Felitti, R.; Bhagirath, A.Y.; Medapati, M.R.; Lai, C.; Cunha, R.S.Antibacterial Properties of Chitosan Nanoparticles and Propolis Associated with Calcium Hydroxide againstSingle- and Multispecies Biofilms: An In Vitro and In Situ Study. J. Endod. 2017, 43, 1332–1336. [CrossRef][PubMed]

135. Tagelsir, A.; Yassen, G.H.; Gomez, G.F.; Gregory, R.L. Effect of Antimicrobials Used in RegenerativeEndodontic Procedures on 3-week-old Enterococcus faecalis Biofilm. J. Endod. 2016, 42, 258–262. [CrossRef][PubMed]

136. Pankajakshan, D.; Albuquerque, M.T.; Evans, J.D.; Kamocka, M.M.; Gregory, R.L.; Bottino, M.C. TripleAntibiotic Polymer Nanofibers for Intracanal Drug Delivery: Effects on Dual Species Biofilm and CellFunction. J. Endod. 2016, 42, 1490–1495. [CrossRef] [PubMed]

137. Chen, J.E.; Nurbakhsh, B.; Layton, G.; Bussmann, M.; Kishen, A. Irrigation dynamics associated with positivepressure, apical negative pressure and passive ultrasonic irrigations: A computational fluid dynamicsanalysis. Aust. Endod. J. 2014, 40, 54–60. [CrossRef] [PubMed]

138. Guerreiro-Tanomaru, J.M.; Chavez-Andrade, G.M.; de Faria-Junior, N.B.; Watanabe, E.; Tanomaru-Filho, M.Effect of Passive Ultrasonic Irrigation on Enterococcus faecalis from Root Canals: An Ex Vivo Study.Br. Dent. J. 2015, 26, 342–346. [CrossRef] [PubMed]

139. Lauterborn, W.; Ohl, C.D. Cavitation bubble dynamics. Ultrason. Sonochem. 1997, 4, 65–75. [CrossRef]140. Layton, G.; Wu, W.I.; Selvaganapathy, P.R.; Friedman, S.; Kishen, A. Fluid Dynamics and Biofilm Removal

Generated by Syringe-delivered and 2 Ultrasonic-assisted Irrigation Methods: A Novel ExperimentalApproach. J. Endod. 2015, 41, 884–889. [CrossRef] [PubMed]

141. Mozo, S.; Llena, C.; Forner, L. Review of ultrasonic irrigation in endodontics: Increasing action of irrigatingsolutions. Med. Oral Patol. Oral Cir. Bucal 2012, 17, e512–e516. [CrossRef] [PubMed]

142. Nair, P.N.; Henry, S.; Cano, V.; Vera, J. Microbial status of apical root canal system of human mandibular firstmolars with primary apical periodontitis after “one-visit” endodontic treatment. Oral Surg. Oral Med. OralPathol. Oral Radiol. Endod. 2005, 99, 231–252. [CrossRef] [PubMed]

143. Vera, J.; Siqueira, J.F., Jr.; Ricucci, D.; Loghin, S.; Fernandez, N.; Flores, B.; Cruz, A.G. One- versus two-visitendodontic treatment of teeth with apical periodontitis: A histobacteriologic study. J. Endod. 2012, 38,1040–1052. [CrossRef] [PubMed]

144. De Groot, S.D.; Verhaagen, B.; Versluis, M.; Wu, M.K.; Wesselink, P.R.; van der Sluis, L.W. Laser-activatedirrigation within root canals: Cleaning efficacy and flow visualization. Int. Endod. J. 2009, 42, 1077–1083.[CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1748 21 of 21

145. Halford, A.; Ohl, C.D.; Azarpazhooh, A.; Basrani, B.; Friedman, S.; Kishen, A. Synergistic effect ofmicrobubble emulsion and sonic or ultrasonic agitation on endodontic biofilm in vitro. J. Endod. 2012,38, 1530–1534. [CrossRef] [PubMed]

146. Ng, Y.L.; Mann, V.; Rahbaran, S.; Lewsey, J.; Gulabivala, K. Outcome of primary root canal treatment:Systematic review of the literature—Part 1. Effects of study characteristics on probability of success.Int. Endod. J. 2007, 40, 921–939. [CrossRef] [PubMed]

147. Ng, Y.L.; Mann, V.; Rahbaran, S.; Lewsey, J.; Gulabivala, K. Outcome of primary root canal treatment:Systematic review of the literature—Part 2. Influence of clinical factors. Int. Endod. J. 2008, 41, 6–31.[CrossRef] [PubMed]

148. Ng, Y.L.; Mann, V.; Gulabivala, K. Tooth survival following non-surgical root canal treatment: A systematicreview of the literature. Int. Endod. J. 2010, 43, 171–189. [CrossRef] [PubMed]

149. Ng, Y.L.; Mann, V.; Gulabivala, K. A prospective study of the factors affecting outcomes of nonsurgical rootcanal treatment: Part 1: Periapical health. Int. Endod. J. 2011, 44, 583–609. [CrossRef] [PubMed]

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